Computationally optimized broadly reactive antigens for h5n1 and h1n1 influenza viruses

ABSTRACT

Described herein is the generation of optimized H5N1 and H1N1 influenza HA polypeptides for eliciting a broadly reactive immune response to influenza virus isolates. The optimized HA polypeptides were developed through a series of HA protein alignments, and subsequent generation of consensus sequences, based on H5N1 and H1N1 influenza isolates. Provided herein are optimized H5N1 and H1N1 influenza HA polypeptides, and compositions, fusion proteins and VLPs comprising the HA polypeptides. Further provided are codon-optimized nucleic acid sequences encoding the HA polypeptides. Methods of eliciting an immune response against influenza virus in a subject are also provided by the present disclosure.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/617,815, filed Mar. 30, 2012, which is herein incorporated byreference in its entirety.

FIELD

This disclosure concerns optimized influenza hemagglutinin proteins thatelicit broadly reactive immune responses to H5N1 and H1N1 influenzaviruses, and their use as vaccines.

BACKGROUND

Influenza virus is a member of Orthomyxoviridae family. There are threesubtypes of influenza viruses, designated influenza A, influenza B, andinfluenza C. The influenza virion contains a segmented negative-senseRNA genome, which encodes the following proteins: hemagglutinin (HA),neuraminidase (NA), matrix (M1), proton ion-channel protein (M2),nucleoprotein (NP), polymerase basic protein 1 (PB1), polymerase basicprotein 2 (PB2), polymerase acidic protein (PA), and nonstructuralprotein 2 (NS2). The HA, NA, M1, and M2 proteins are membraneassociated, whereas NP, PB1, PB2, PA, and NS2 are nucleocapsidassociated proteins. The M1 protein is the most abundant protein ininfluenza particles. The HA and NA proteins are envelope glycoproteins,responsible for virus attachment and penetration of the viral particlesinto the cell, and the sources of the major immunodominant epitopes forvirus neutralization and protective immunity. Both HA and NA proteinsare considered the most important components for prophylactic influenzavaccines.

Each year, seasonal influenza causes over 300,000 hospitalizations and36,000 deaths in the U.S. alone (Simonsen et al., Lancet Infect Dis7:658-66, 2007). The emergence of the novel H1N1 influenza virus in 2009demonstrated how quickly a new influenza pandemic can sweep across theworld.

There are currently two influenza vaccine approaches licensed in theUnited States—the inactivated, split vaccine and the live-attenuatedvirus vaccine. The inactivated vaccines can efficiently induce humoralimmune responses but generally only poor cellular immune responses. Livevirus vaccines cannot be administered to immunocompromised or pregnantpatients due to their increased risk of infection. Thus, a need existsfor a broadly protective influenza virus vaccine.

SUMMARY

Disclosed herein is the generation of computationally-optimized H5N1 andH1N1 influenza HA polypeptides for eliciting a broadly reactive immuneresponse to influenza virus. The optimized HA polypeptides weredeveloped through a series of HA protein alignments, and subsequentgeneration of consensus sequences based on selected H5N1 and H1N1influenza virus isolates.

Provided herein are recombinant influenza HA polypeptides having anoptimized amino acid sequence for eliciting a broadly reactive immuneresponse against H5N1 or H1N1 influenza, wherein the HA polypeptidecomprises or consists of the amino acid sequence of SEQ ID NO: 1 or SEQID NO: 2. In some embodiments, the influenza HA polypeptide lacks theN-terminal methionine residue.

Isolated nucleic acid molecules and vectors encoding the recombinant HApolypeptides are also provided by the present disclosure. Furtherprovided are isolated cells comprising such vectors.

Also provided are influenza virus-like particles (VLPs) and fusionproteins comprising the optimized HA polypeptides disclosed herein.

Further provided are compositions that include the optimized influenzaHA polypeptides, fusion proteins or VLPs disclosed herein in apharmaceutically acceptable carrier. Methods of eliciting an immuneresponse against influenza virus in a subject by administering thedisclosed compositions, fusion proteins or VLPs are also provided by thepresent disclosure.

Also provided are methods of immunizing a subject against influenzavirus by administering to the subject a composition comprising a VLPthat contains an optimized HA polypeptide.

The foregoing and other objects, features, and advantages of theinvention will become more apparent from the following detaileddescription, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic summarizing the process for generating a COBRA HAsequence using 426 human H5N1 isolates from clades 0, 1, 2.1, 2.2, 2.3and 7, which is referred to herein as the “PATH H5N1 COBRA” HA.

FIG. 2 is a schematic summarizing the process for generating a COBRA HAsequence using 205 human and swine H1N1 influenza virus isolates, whichis referred to herein as the “PATH H1N1 COBRA” HA.

FIG. 3 is a graph showing HAI titers against clade 1 and clade 2challenge strains following vaccination with VLPs containing the PATHH5N1 COBRA HA sequence (SEQ ID NO: 1), VLPs containing a human clade 2influenza virus COBRA HA sequence (Human COBRA-2), or VLPs containingWhooper Swan (A/Whooper Swan/Mongolia/244/2005) influenza virus HA.Vaccinations (3 μg) were performed at 0 and 3 weeks with adjuvant(Imject™).

FIG. 4 is a graph showing body weight of animals vaccinated with VLPscontaining the PATH H5N1 COBRA HA sequence (SEQ ID NO: 1), VLPscontaining the Human COBRA-2 HA sequence, or Whooper Swan VLPs andsubsequently challenged with 5000 PFU Indonesia clade 2.1 virus(A/Indonesia/5/2005). Vaccinations (3 μg) were performed at 0 and 3weeks with adjuvant (Imject™); virus challenge occurred during week 5.

FIG. 5 is a graph showing body weight of animals vaccinated with VLPscontaining the PATH H5N1 COBRA HA sequence (SEQ ID NO: 1) andsubsequently challenged with 5000 PFU Vietnam clade 1 virus(A/Vietnam/1203/2004). Vaccinations (3 μg) were performed at 0 and 3weeks with adjuvant (Imject™); virus challenge occurred during week 5.

FIG. 6 is a graph showing body weight of animals vaccinated with VLPscontaining the PATH H5N1 COBRA HA sequence (SEQ ID NO: 1), VLPscontaining the Human COBRA-2 HA sequence, or Whooper Swan VLPs, andsubsequently challenged with Vietnam clade 1 virus. A single vaccination(3 μg) was performed at week 0 with adjuvant (Imject™), followed byvirus challenge during week 4.

FIG. 7 is a graph showing percent survival of animals vaccinated withVLPs containing the PATH H5N1 COBRA HA sequence (SEQ ID NO: 1), VLPscontaining the Human COBRA-2 HA sequence, or Whooper Swan VLPs, andsubsequently challenged with Vietnam clade 1 virus. A single vaccination(3 μg) was performed at week 0 without adjuvant, followed by viruschallenge during week 4.

FIG. 8 is a graph showing body weight of animals vaccinated with VLPscontaining the PATH H5N1 COBRA HA sequence (SEQ ID NO: 1), VLPscontaining the Human COBRA-2 HA sequence, or Whooper Swan VLPs, andsubsequently challenged with Vietnam clade 1 virus. A single vaccinationat a dose of 0.6 μg was performed at week 0 with adjuvant (Imject™),followed by virus challenge during week 4.

SEQUENCE LISTING

The amino acid sequences listed in the accompanying sequence listing areshown using three letter code for amino acids, as defined in 37 C.F.R.1.822. The Sequence Listing is submitted as an ASCII text file, createdon Feb. 7, 2013, 10.0 KB, which is incorporated by reference herein. Inthe accompanying sequence listing:

SEQ ID NO: 1 is a COBRA amino acid sequence for H5N1 influenza HA (“PATHH5N1 COBRA”).

SEQ ID NO: 2 is a COBRA amino acid sequence for H1N1 influenza HA (“PATHH1N1 COBRA”).

DETAILED DESCRIPTION I. Abbreviations

COBRA: computationally optimized broadly reactive antigen

HA: hemagglutinin

HAI: hemagglutination inhibition

HRP: horseradish peroxidase

M1: matrix protein 1

NA: neuraminidase

PFU: plaque form unit

VLP: virus-like particle

II. Terms and Methods

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

In order to facilitate review of the various embodiments of thedisclosure, the following explanations of specific terms are provided:

Adjuvant: A substance or vehicle that non-specifically enhances theimmune response to an antigen. Adjuvants can include a suspension ofminerals (alum, aluminum hydroxide, or phosphate) on which antigen isadsorbed; or water-in-oil emulsion in which antigen solution isemulsified in mineral oil (for example, Freund's incomplete adjuvant),sometimes with the inclusion of killed mycobacteria (Freund's completeadjuvant) to further enhance antigenicity. Immunostimulatoryoligonucleotides (such as those including a CpG motif) can also be usedas adjuvants (for example, see U.S. Pat. Nos. 6,194,388; 6,207,646;6,214,806; 6,218,371; 6,239,116; 6,339,068; 6,406,705; and 6,429,199).Adjuvants also include biological molecules, such as costimulatorymolecules. Exemplary biological adjuvants include IL-2, RANTES, GM-CSF,TNF-α, IFN-γ, G-CSF, LFA-3, CD72, B7-1, B7-2, OX-40L and 41 BBL.

Administer: As used herein, administering a composition to a subjectmeans to give, apply or bring the composition into contact with thesubject. Administration can be accomplished by any of a number ofroutes, such as, for example, topical, oral, subcutaneous,intramuscular, intraperitoneal, intravenous, intrathecal andintradermal.

Antibody: An immunoglobulin molecule produced by B lymphoid cells with aspecific amino acid sequence. Antibodies are evoked in humans or otheranimals by a specific antigen (immunogen). Antibodies are characterizedby reacting specifically with the antigen in some demonstrable way,antibody and antigen each being defined in terms of the other.“Eliciting an antibody response” refers to the ability of an antigen orother molecule to induce the production of antibodies.

Antigen: A compound, composition, or substance that can stimulate theproduction of antibodies or a T-cell response in an animal, includingcompositions that are injected or absorbed into an animal. An antigenreacts with the products of specific humoral or cellular immunity,including those induced by heterologous immunogens. In some embodimentsof the disclosed compositions and methods, the antigen is an influenzaHA protein.

Codon-optimized: A “codon-optimized” nucleic acid refers to a nucleicacid sequence that has been altered such that the codons are optimal forexpression in a particular system (such as a particular species or groupof species). For example, a nucleic acid sequence can be optimized forexpression in mammalian cells. Codon optimization does not alter theamino acid sequence of the encoded protein.

Fusion protein: A protein generated by expression of a nucleic acidsequence engineered from nucleic acid sequences encoding at least aportion of two different (heterologous) proteins. To create a fusionprotein, the nucleic acid sequences must be in the same reading frameand contain no internal stop codons. For example, a fusion protein caninclude an influenza HA fused to a heterologous protein.

Hemagglutinin (HA): An influenza virus surface glycoprotein. HA mediatesbinding of the virus particle to a host cells and subsequent entry ofthe virus into the host cell. The nucleotide and amino acid sequences ofnumerous influenza HA proteins are known in the art and are publicallyavailable, such as through the NCBI Influenza Virus Resource database(Bao et al., J Virol 82:596-601, 2008). HA (along with NA) is one of thetwo major influenza virus antigenic determinants.

Immune response: A response of a cell of the immune system, such as aB-cell, T-cell, macrophage or polymorphonucleocyte, to a stimulus suchas an antigen or vaccine. An immune response can include any cell of thebody involved in a host defense response, including for example, anepithelial cell that secretes an interferon or a cytokine. An immuneresponse includes, but is not limited to, an innate immune response orinflammation. As used herein, a protective immune response refers to animmune response that protects a subject from infection (preventsinfection or prevents the development of disease associated withinfection). Methods of measuring immune responses are well known in theart and include, for example, measuring proliferation and/or activity oflymphocytes (such as B or T cells), secretion of cytokines orchemokines, inflammation, antibody production and the like.

Immunogen: A compound, composition, or substance which is capable, underappropriate conditions, of stimulating an immune response, such as theproduction of antibodies or a T-cell response in an animal, includingcompositions that are injected or absorbed into an animal. As usedherein, an “immunogenic composition” is a composition comprising animmunogen (such as an HA polypeptide).

Immunize: To render a subject protected from an infectious disease, suchas by vaccination.

Influenza virus: A segmented negative-strand RNA virus that belongs tothe Orthomyxoviridae family. There are three types of influenza viruses,A, B and C. Influenza A viruses infect a wide variety of birds andmammals, including humans, horses, marine mammals, pigs, ferrets, andchickens. In animals, most influenza A viruses cause mild localizedinfections of the respiratory and intestinal tract. However, highlypathogenic influenza A strains, such as H5N1, cause systemic infectionsin poultry in which mortality may reach 100%. In 2009, H1N1 influenzawas the most common cause of human influenza. A new strain ofswine-origin H1N1 emerged in 2009 and was declared pandemic by the WorldHealth Organization. This strain was referred to as “swine flu.” H1N1influenza A viruses were also responsible for the Spanish flu pandemicin 1918, the Fort Dix outbreak in 1976, and the Russian flu epidemic in1977-1978.

Isolated: An “isolated” biological component (such as a nucleic acid,protein or virus) has been substantially separated or purified away fromother biological components (such as cell debris, or other proteins ornucleic acids). Biological components that have been “isolated” includethose components purified by standard purification methods. The termalso embraces recombinant nucleic acids, proteins or viruses (or VLPs),as well as chemically synthesized nucleic acids or peptides.

Linker: One or more amino acids that serve as a spacer between twopolypeptides of a fusion protein.

Matrix (M1) protein: An influenza virus structural protein found withinthe viral envelope. M1 is thought to function in assembly and budding.

Neuraminidase (NA): An influenza virus membrane glycoprotein. NA isinvolved in the destruction of the cellular receptor for the viral HA bycleaving terminal sialic acid residues from carbohydrate moieties on thesurfaces of infected cells. NA also cleaves sialic acid residues fromviral proteins, preventing aggregation of viruses. NA (along with HA) isone of the two major influenza virus antigenic determinants.

Operably linked: A first nucleic acid sequence is operably linked with asecond nucleic acid sequence when the first nucleic acid sequence isplaced in a functional relationship with the second nucleic acidsequence. For instance, a promoter is operably linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence. Generally, operably linked DNA sequences are contiguousand, where necessary to join two protein-coding regions, in the samereading frame.

Optimized influenza HA protein: As used herein, “optimized influenza HAprotein” refers to an HA protein consensus sequence generated bysequence alignments of H5N1 and H1N1 influenza viruses isolates (asdescribed in Examples 1 and 2 below). Nucleotide sequences encodingoptimized HA proteins are further optimized for expression in mammaliancells via codon-optimization and RNA optimization (such as to increaseRNA stability). Optimized influenza HA proteins disclosed herein (andset forth herein as SEQ ID NO: 1 and SEQ ID NO: 2) are also referred toas “COBRA” (computationally-optimized broadly reactive antigen)sequences. Optimized HA polypeptides are designed to elicit broadlyreactive immune responses in a subject. In the context of the presentdisclosure, “broadly reactive” means the protein sequence elicits animmune response in a subject that is sufficient to inhibit, neutralizeor prevent infection of a broad range of influenza viruses (such as mostor all influenza viruses within a specific subtype). In some instances,the optimized influenza HA protein is capable of eliciting an immuneresponse, such as a protective immune response, against most or all H5N1influenza virus isolates, or most or all H1N1 influenza virus isolates.

Outbreak: As used herein, an influenza virus “outbreak” refers to acollection of virus isolates from within a single country in a givenyear.

Pharmaceutically acceptable vehicles: The pharmaceutically acceptablecarriers (vehicles) useful in this disclosure are conventional.Remington's Pharmaceutical Sciences, by E. W. Martin, Mack PublishingCo., Easton, Pa., 15th Edition (1975), describes compositions andformulations suitable for pharmaceutical delivery of one or moretherapeutic compositions, such as one or more influenza vaccines, andadditional pharmaceutical agents.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (for example, powder, pill, tablet, orcapsule forms), conventional non-toxic solid carriers can include, forexample, pharmaceutical grades of mannitol, lactose, starch, ormagnesium stearate. In addition to biologically-neutral carriers,pharmaceutical compositions to be administered can contain minor amountsof non-toxic auxiliary substances, such as wetting or emulsifyingagents, preservatives, and pH buffering agents and the like, for examplesodium acetate or sorbitan monolaurate.

Polypeptide: A polymer in which the monomers are amino acid residueswhich are joined together through amide bonds. When the amino acids arealpha-amino acids, either the L-optical isomer or the D-optical isomercan be used. The terms “polypeptide” or “protein” as used herein areintended to encompass any amino acid sequence and include modifiedsequences such as glycoproteins. The term “polypeptide” is specificallyintended to cover naturally occurring proteins, as well as those whichare recombinantly or synthetically produced. The term “residue” or“amino acid residue” includes reference to an amino acid that isincorporated into a protein, polypeptide, or peptide.

Preventing, treating or ameliorating a disease: “Preventing” a diseaserefers to inhibiting the full development of a disease. “Treating”refers to a therapeutic intervention that ameliorates a sign or symptomof a disease or pathological condition after it has begun to develop.“Ameliorating” refers to the reduction in the number or severity ofsigns or symptoms of a disease.

Promoter: A promoter is an array of nucleic acid control sequences whichdirect transcription of a nucleic acid. A promoter includes necessarynucleic acid sequences near the start site of transcription. A promoteralso optionally includes distal enhancer or repressor elements. A“constitutive promoter” is a promoter that is continuously active and isnot subject to regulation by external signals or molecules. In contrast,the activity of an “inducible promoter” is regulated by an externalsignal or molecule (for example, a transcription factor). In someembodiments herein, the promoter is a CMV promoter.

Purified: The term “purified” does not require absolute purity; rather,it is intended as a relative term. Thus, for example, a purifiedpeptide, protein, virus, VLP or other active compound is one that isisolated in whole or in part from naturally associated proteins andother contaminants. In certain embodiments, the term “substantiallypurified” refers to a peptide, protein, virus, VLP or other activecompound that has been isolated from a cell, cell culture medium, orother crude preparation and subjected to fractionation to remove variouscomponents of the initial preparation, such as proteins, cellulardebris, and other components.

Recombinant: A recombinant nucleic acid, protein, virus or VLP is onethat has a sequence that is not naturally occurring or has a sequencethat is made by an artificial combination of two otherwise separatedsegments of sequence. This artificial combination is often accomplishedby chemical synthesis or by the artificial manipulation of isolatedsegments of nucleic acids, for example, by genetic engineeringtechniques.

Subject: Living multi-cellular vertebrate organisms, a category thatincludes both human and non-human mammals, such as non-human primates.

Therapeutically effective amount: A quantity of a specified agentsufficient to achieve a desired effect in a subject being treated withthat agent. For example, this may be the amount of an influenza virusvaccine useful for eliciting an immune response in a subject and/or forpreventing infection or disease caused by influenza virus. Ideally, inthe context of the present disclosure, a therapeutically effectiveamount of an influenza vaccine is an amount sufficient to increaseresistance to, prevent, ameliorate, and/or treat infection caused byinfluenza virus in a subject without causing a substantial cytotoxiceffect in the subject. The effective amount of an influenza vaccineuseful for increasing resistance to, preventing, ameliorating, and/ortreating infection in a subject will be dependent on, for example, thesubject being treated, the manner of administration of the therapeuticcomposition and other factors.

Transformed: A transformed cell is a cell into which has been introduceda nucleic acid molecule by molecular biology techniques. As used herein,the term transformation encompasses all techniques by which a nucleicacid molecule might be introduced into such a cell, includingtransfection with viral vectors, transformation with plasmid vectors,and introduction of naked DNA by electroporation, lipofection, andparticle gun acceleration.

Vaccine: A preparation of immunogenic material capable of stimulating animmune response, administered for the prevention, amelioration, ortreatment of disease, such as an infectious disease. The immunogenicmaterial may include, for example, attenuated or killed microorganisms(such as attenuated viruses), or antigenic proteins (including VLPs),peptides or DNA derived from them. Vaccines may elicit both prophylactic(preventative) and therapeutic responses. Methods of administration varyaccording to the vaccine, but may include inoculation, ingestion,inhalation or other forms of administration. Inoculations can bedelivered by any of a number of routes, including parenteral, such asintravenous, subcutaneous or intramuscular. Vaccines may be administeredwith an adjuvant to boost the immune response.

Vector: A vector is a nucleic acid molecule allowing insertion offoreign nucleic acid without disrupting the ability of the vector toreplicate and/or integrate in a host cell. A vector can include nucleicacid sequences that permit it to replicate in a host cell, such as anorigin of replication. An insertional vector is capable of insertingitself into a host nucleic acid. A vector can also include one or moreselectable marker genes and other genetic elements. An expression vectoris a vector that contains the necessary regulatory sequences to allowtranscription and translation of an inserted gene or genes. In someembodiments of the present disclosure, the vector encodes an influenzaHA, NA or M1 protein. In some embodiments, the vector is the pTR600expression vector (U.S. Patent Application Publication No. 2002/0106798;Ross et al., Nat Immunol. 1(2):102-103, 2000; Green et al., Vaccine20:242-248, 2001).

Virus-like particle (VLP): Virus particles made up of one of more viralstructural proteins, but lacking the viral genome. Because VLPs lack aviral genome, they are non-infectious. In addition, VLPs can often beproduced by heterologous expression and can be easily purified. MostVLPs comprise at least a viral core protein that drives budding andrelease of particles from a host cell. One example of such a coreprotein is influenza M1. In some embodiments herein, an influenza VLPcomprises the HA, NA and/or M1 proteins. Influenza VLPs can be producedby transfection of host cells with plasmids encoding the HA and NAproteins, and optionally the M1 protein. After incubation of thetransfected cells for an appropriate time to allow for proteinexpression (such as for approximately 72 hours), VLPs can be isolatedfrom cell culture supernatants. Example 4 provides an exemplary protocolfor purifying influenza VLPs from cell supernatants. In this example,VLPs are isolated by low speed centrifugation (to remove cell debris),vacuum filtration and ultracentrifugation through 20% glycerol. Othermethods of producing influenza VLPs are known in the art (see, forexample, U.S. Patent Application Publication Nos. 2006/0263804;2008/0031895; 2010/0166769; and 2010/0239610).

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. “Comprising A or B” means including A, or B, or Aand B. It is further to be understood that all base sizes or amino acidsizes, and all molecular weight or molecular mass values, given fornucleic acids or polypeptides are approximate, and are provided fordescription. Although methods and materials similar or equivalent tothose described herein can be used in the practice or testing of thepresent disclosure, suitable methods and materials are described below.All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including explanations ofterms, will control. In addition, the materials, methods, and examplesare illustrative only and not intended to be limiting.

III. Overview of Several Embodiments

Disclosed herein is the generation of computationally-optimized H5N1 andH1N1 influenza HA polypeptides for eliciting a broadly reactive immuneresponse to influenza virus. The optimized HA polypeptides weredeveloped through a series of HA protein alignments, and subsequentgeneration of consensus sequences based on selected H5N1 and H1N1influenza virus isolates. The methods used to generate the optimized HAconsensus sequences are described in Examples 1 and 2, and shown inFIGS. 1 and 2. The amino acid sequences of 2 specific HA polypeptidesare set forth herein as SEQ ID NO: 1 (H5N1) and SEQ ID NO: 2 (H1N1). TheH5N1 sequence was generated using 426 human H5N1 isolates from clades 0,1, 2.1, 2.2, 2.3 and 7, and is referred to herein as the “PATH H5N1COBRA” HA. The H1N1 sequence was generated using 205 human and swineH1N1 influenza virus isolates, and is referred to herein as the “PATHH1N1 COBRA” HA.

Provided herein are recombinant influenza HA polypeptides having anoptimized amino acid sequence for eliciting a broadly reactive immuneresponse against H5N1 or H1N1 influenza. In some embodiments, the aminoacid sequence of the HA polypeptide comprises or consists of the aminoacid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

In other embodiments, the amino acid sequence of the HA polypeptidecomprises or consists of the amino acid sequence of residues 2-568 ofSEQ ID NO: 1, or residues 2-566 of SEQ ID NO: 2.

Further provided are isolated nucleic acid molecules encoding arecombinant influenza HA polypeptide disclosed herein. In someembodiments, the nucleic acid molecule is codon-optimized for expressionin mammalian cells. The nucleic acid molecule is optionally furtheroptimized for RNA stability.

Vectors comprising the nucleic acid molecules encoding recombinant HApolypeptides are also provided by the present disclosure. The vector canbe any suitable vector for expression of the HA polypeptide, such as amammalian expression vector. In particular examples, the vector is thepTR600 expression vector (U.S. Patent Application Publication No.2002/0106798, herein incorporated by reference; Ross et al., NatImmunol. 1(2):102-103, 2000; Green et al., Vaccine 20:242-248, 2001).

In some examples, the vector includes a promoter operably linked to thenucleic acid sequence encoding the HA polypeptide. In particularexamples, the promoter is a CMV promoter.

Also provided are isolated cells comprising the disclosed vectors. Insome cases, the cell is any suitable cell type for production andexpression of VLPs, such as a mammalian cell.

Further provided are influenza VLPs comprising an optimized HApolypeptide disclosed herein. The influenza VLPs can further include anyadditional influenza proteins necessary to form the virus particle. Insome embodiments, the influenza VLPs further include influenzaneuraminidase (NA) protein, influenza matrix (M1) protein, or both.

Also provided are influenza VLPs comprising an influenza HA polypeptidedisclosed herein, produced by transfecting a host cell with a vectorencoding the HA polypeptide, a vector encoding an influenza NA proteinand a vector encoding an influenza M1 protein under conditionssufficient to allow for expression of the HA, M1 and NA proteins.

Fusion proteins comprising an optimized influenza HA polypeptide arefurther provided by the present disclosure.

Also provided herein are compositions comprising an optimized influenzaHA protein as disclosed herein, or a fusion protein or VLP comprisingthe optimized influenza HA protein. In some embodiments, thecompositions further comprise a pharmaceutically acceptable carrierand/or an adjuvant. For example, the adjuvant can be alum, Freund'scomplete adjuvant, a biological adjuvant or immunostimulatoryoligonucleotides (such as CpG oligonucleotides).

Further provided is a method of eliciting an immune response toinfluenza virus in a subject by administering an optimized influenza HAprotein, a fusion protein comprising an optimized influenza HA, VLPscontaining an optimized influenza HA, or compositions thereof, asdisclosed herein. In some embodiments, the influenza virus is an H5N1 orH1N1 influenza virus. In some embodiments, the HA protein, HA fusionprotein or VLP can be administered using any suitable route ofadministration, such as, for example, intramuscular, intranasal or oral.In some embodiments, the HA protein, fusion protein or VLP isadministered as a composition further comprising a pharmaceuticallyacceptable carrier and/or an adjuvant. For example, the adjuvant can bealum, Freund's complete adjuvant, a biological adjuvant orimmunostimulatory oligonucleotides (such as CpG oligonucleotides).

Also provided is a method of immunizing a subject against influenzavirus by administering to the subject VLPs containing an optimizedinfluenza HA protein disclosed herein, or administering a compositionthereof. In some embodiments of the method, the composition furthercomprises a pharmaceutically acceptable carrier and/or an adjuvant. Forexample, the adjuvant can be alum, Freund's complete adjuvant, abiological adjuvant or immunostimulatory oligonucleotides (such as CpGoligonucleotides). In some embodiments, the VLPs (or compositionsthereof) are administered intramuscularly.

In some embodiments of the methods of eliciting an immune response orimmunizing a subject, the subject is administered about 1 to about 25 μgof the VLPs containing an optimized HA protein. In particular examples,the subject is administered about 5 to about 20 μg of the VLPs, or about10 to about 15 μg of the VLPs. In one specific non-limiting example, thesubject is administered about 15 μg of the VLPs. However, one of skillin the art is capable of determining a therapeutically effective amount(for example an amount that provides protection against H5N1 or H1N1influenza virus infection) of VLPs to administer to a subject.

IV. Influenza

Influenza viruses are segmented negative-strand RNA viruses that belongto the Orthomyxoviridae family. There are three types of Influenzaviruses, A, B and C. Influenza A viruses infect a wide variety of birdsand mammals, including humans, horses, marine mammals, pigs, ferrets,and chickens. In animals, most influenza A viruses cause mild localizedinfections of the respiratory and intestinal tract. However, highlypathogenic influenza A strains, such as H5N1, cause systemic infectionsin poultry in which mortality may reach 100%. Animals infected withinfluenza A often act as a reservoir for the influenza viruses andcertain subtypes have been shown to cross the species barrier to humans.

Influenza A viruses can be classified into subtypes based on allelicvariations in antigenic regions of two genes that encode surfaceglycoproteins, namely, hemagglutinin (HA) and neuraminidase (NA) whichare required for viral attachment and cellular release. Currently,sixteen subtypes of HA (H1-H16) and nine NA (N1-N9) antigenic variantsare known for influenza A virus. Previously, only three subtypes wereknown to circulate in humans (H1N1, H1N2, and H3N2). However, in recentyears, the pathogenic H5N1 subtype of avian influenza A has beenreported to cross the species barrier and infect humans as documented inHong Kong in 1997 and 2003, leading to the death of several patients.

In 2009, H1N1 influenza was the most common cause of human influenza. Anew strain of swine-origin H1N1 emerged in 2009 and was declaredpandemic by the World Health Organization. This strain was referred toas “swine flu.” H1N1 influenza A viruses were also responsible for theSpanish flu pandemic in 1918, the Fort Dix outbreak in 1976, and theRussian flu epidemic in 1977-1978.

The influenza virus segmented genome contains eight negative-sense RNA(nsRNA) gene segments (PB2, PB1, PA, NP, M, NS, HA and NA) that encodeat least ten polypeptides, including RNA-directed RNA polymeraseproteins (PB2, PB 1 and PA), nucleoprotein (NP), neuraminidase (NA),hemagglutinin (subunits HA1 and HA2), the matrix proteins (M1 and M2)and the non-structural proteins (NS1 and NS2) (Krug et al., In “TheInfluenza Viruses,” R. M. Krug, ed., Plenum Press, N.Y., 1989, pp. 89152).

Influenza virus' ability to cause widespread disease is due to itsability to evade the immune system by undergoing antigenic change, whichis believed to occur when a host is infected simultaneously with both ananimal influenza virus and a human influenza virus. During mutation andreassortment in the host, the virus may incorporate an HA and/or NAsurface protein gene from another virus into its genome, therebyproducing a new influenza subtype and evading the immune system.

HA is a viral surface glycoprotein generally comprising approximately560 amino acids and representing 25% of the total virus protein. It isresponsible for adhesion of the viral particle to, and its penetrationinto, a host cell in the early stages of infection. Cleavage of thevirus HA0 precursor into the HA1 and HA2 sub-fragments is a necessarystep in order for the virus to infect a cell. Thus, cleavage is requiredin order to convert new virus particles in a host cell into virionscapable of infecting new cells. Cleavage is known to occur duringtransport of the integral HA0 membrane protein from the endoplasmicreticulum of the infected cell to the plasma membrane. In the course oftransport, hemagglutinin undergoes a series of co- andpost-translational modifications including proteolytic cleavage of theprecursor HA into the amino-terminal fragment HA1 and the carboxyterminal HA2. One of the primary difficulties in growing influenzastrains in primary tissue culture or established cell lines arises fromthe requirement for proteolytic cleavage activation of the influenzahemagglutinin in the host cell.

Although it is known that an uncleaved HA can mediate attachment of thevirus to its neuraminic acid-containing receptors on a cell surface, itis not capable of the next step in the infectious cycle, which isfusion. It has been reported that exposure of the hydrophobic aminoterminus of HA2 by cleavage is required so that it can be inserted intothe target cell, thereby forming a bridge between virus and target cellmembrane. This process is followed by fusion of the two membranes andentry of the virus into the target cell.

Proteolytic activation of HA involves cleavage at an arginine residue bya trypsin-like endoprotease, which is often an intracellular enzyme thatis calcium dependent and has a neutral pH optimum. Since the activatingproteases are cellular enzymes, the infected cell type determineswhether the HA is cleaved. The HA of the mammalian influenza viruses andthe nonpathogenic avian influenza viruses are susceptible to proteolyticcleavage only in a restricted number of cell types. On the other hand,HA of pathogenic avian viruses among the H5 and H7 subtypes are cleavedby proteases present in a broad range of different host cells. Thus,there are differences in host range resulting from differences inhemagglutinin cleavability which are correlated with the pathogenicproperties of the virus.

Neuraminidase (NA) is a second membrane glycoprotein of the influenzaviruses. The presence of viral NA has been shown to be important forgenerating a multi-faceted protective immune response against aninfecting virus. For most influenza A viruses, NA is 413 amino acids inlength, and is encoded by a gene of 1413 nucleotides. Nine different NAsubtypes have been identified in influenza viruses (N1, N2, N3, N4, N5,N6, N7, N8 and N9), all of which have been found among wild birds. NA isinvolved in the destruction of the cellular receptor for the viral HA bycleaving terminal neuraminic acid (also called sialic acid) residuesfrom carbohydrate moieties on the surfaces of infected cells. NA alsocleaves sialic acid residues from viral proteins, preventing aggregationof viruses. Using this mechanism, it is hypothesized that NA facilitatesrelease of viral progeny by preventing newly formed viral particles fromaccumulating along the cell membrane, as well as by promotingtransportation of the virus through the mucus present on the mucosalsurface. NA is an important antigenic determinant that is subject toantigenic variation.

In addition to the surface proteins HA and NA, influenza virus comprisessix additional internal genes, which give rise to eight differentproteins, including polymerase genes PB 1, PB2 and PA, matrix proteinsM1 and M2, nucleoprotein (NP), and non-structural proteins NS1 and NS2(Horimoto et al., Clin Microbiol Rev. 14(1):129-149, 2001).

In order to be packaged into progeny virions, viral RNA is transportedfrom the nucleus as a ribonucleoprotein (RNP) complex composed of thethree influenza virus polymerase proteins, the nucleoprotein (NP), andthe viral RNA, in association with the influenza virus matrix 1 (M1)protein and nuclear export protein (Marsh et al., J Virol, 82:2295-2304,2008). The M1 protein that lies within the envelope is thought tofunction in assembly and budding. A limited number of M2 proteins areintegrated into the virions (Zebedee, J. Virol. 62:2762-2772, 1988).They form tetramers having H+ ion channel activity, and when activatedby the low pH in endosomes, acidify the inside of the virion,facilitating its uncoating (Pinto et al., Cell 69:517-528, 1992).Amantadine is an anti-influenza drug that prevents viral infection byinterfering with M2 ion channel activity, thus inhibiting virusuncoating.

NS1, a nonstructural protein, has multiple functions, includingregulation of splicing and nuclear export of cellular mRNAs as well asstimulation of translation. The major function of NS1 seems to be tocounteract the interferon activity of the host, since an NS1 knockoutvirus was viable although it grew less efficiently than the parent virusin interferon-nondefective cells (Garcia-Sastre, Virology 252:324-330,1998).

NS2 has been detected in virus particles (Richardson et al., Arch.Virol. 116:69-80, 1991; Yasuda et al., Virology 196:249-255, 1993). Theaverage number of NS2 proteins in a virus particle was estimated to be130-200 molecules. An in vitro binding assay shows directprotein-protein contact between M1 and NS2. NS2-M1 complexes have alsobeen detected by immunoprecipitation in virus-infected cell lysates. TheNS2 protein is thought to play a role in the export of RNP from thenucleus through interaction with M1 protein (Ward et al., Arch. Virol.140:2067-2073, 1995).

V. Influenza VLPs and Administration Thereof

Influenza VLPs comprising an optimized HA (such as the HA having theamino acid sequence set forth as SEQ ID NO: 1 or SEQ ID NO: 2) areprovided herein. The influenza VLPs are generally made up of the HA, NAand M1 proteins. The production of influenza VLPs has been described inthe art and is within the abilities of one of ordinary skill in the art.For example, influenza VLPs can be produced by transfection of hostcells with plasmids encoding the HA, NA and M1 proteins. Afterincubation of the transfected cells for an appropriate time to allow forprotein expression (such as for approximately 72 hours), VLPs can beisolated from cell culture supernatants. Example 4 below provides anexemplary protocol for purifying influenza VLPs from cell supernatants.In this example, VLPs are isolated by low speed centrifugation (toremove cell debris), vacuum filtration and ultracentrifugation through20% glycerol.

The influenza VLPs disclosed herein can be used as influenza vaccines toelicit a protective immune response against H5N1 and H1N1 influenzaviruses.

Influenza VLPs, or compositions thereof, can be administered to asubject by any of the routes normally used for introducing recombinantvirus into a subject. Methods of administration include, but are notlimited to, intradermal, intramuscular, intraperitoneal, parenteral,intravenous, subcutaneous, vaginal, rectal, intranasal, inhalation ororal. Parenteral administration, such as subcutaneous, intravenous orintramuscular administration, is generally achieved by injection.Injectables can be prepared in conventional forms, either as liquidsolutions or suspensions, solid forms suitable for solution orsuspension in liquid prior to injection, or as emulsions. Injectionsolutions and suspensions can be prepared from sterile powders,granules, and tablets of the kind previously described. Administrationcan be systemic or local.

Influenza VLPs, or compositions thereof, are administered in anysuitable manner, such as with pharmaceutically acceptable carriers.Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of pharmaceutical compositions of thepresent disclosure.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like.

Some of the compositions may potentially be administered as apharmaceutically acceptable acid- or base-addition salt, formed byreaction with inorganic acids such as hydrochloric acid, hydrobromicacid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, andphosphoric acid, and organic acids such as formic acid, acetic acid,propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid,malonic acid, succinic acid, maleic acid, and fumaric acid, or byreaction with an inorganic base such as sodium hydroxide, ammoniumhydroxide, potassium hydroxide, and organic bases such as mono-, di-,trialkyl and aryl amines and substituted ethanolamines.

Administration can be accomplished by single or multiple doses. The doseadministered to a subject in the context of the present disclosureshould be sufficient to induce a beneficial therapeutic response in asubject over time, or to inhibit or prevent H5N1 or H1N1 influenza virusinfection. The dose required will vary from subject to subject dependingon the species, age, weight and general condition of the subject, theseverity of the infection being treated, the particular compositionbeing used and its mode of administration. An appropriate dose can bedetermined by one of ordinary skill in the art using only routineexperimentation.

Provided herein are pharmaceutical compositions which include atherapeutically effective amount of the influenza VLPs alone or incombination with a pharmaceutically acceptable carrier. Pharmaceuticallyacceptable carriers include, but are not limited to, saline, bufferedsaline, dextrose, water, glycerol, ethanol, and combinations thereof.The carrier and composition can be sterile, and the formulation suitsthe mode of administration. The composition can also contain minoramounts of wetting or emulsifying agents, or pH buffering agents. Thecomposition can be a liquid solution, suspension, emulsion, tablet,pill, capsule, sustained release formulation, or powder. The compositioncan be formulated as a suppository, with traditional binders andcarriers such as triglycerides. Oral formulations can include standardcarriers such as pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, sodium saccharine, cellulose, and magnesiumcarbonate. Any of the common pharmaceutical carriers, such as sterilesaline solution or sesame oil, can be used. The medium can also containconventional pharmaceutical adjunct materials such as, for example,pharmaceutically acceptable salts to adjust the osmotic pressure,buffers, preservatives and the like. Other media that can be used withthe compositions and methods provided herein are normal saline andsesame oil.

The influenza VLPs described herein can be administered alone or incombination with other therapeutic agents to enhance antigenicity. Forexample, the influenza VLPs can be administered with an adjuvant, suchas Freund incomplete adjuvant or Freund's complete adjuvant.

Optionally, one or more cytokines, such as IL-2, IL-6, IL-12, RANTES,GM-CSF, TNF-α, or IFN-γ, one or more growth factors, such as GM-CSF orG-CSF; one or more molecules such as OX-40L or 41 BBL, or combinationsof these molecules, can be used as biological adjuvants (see, forexample, Salgaller et al., 1998, J. Surg. Oncol. 68(2):122-38; Lotze etal., 2000, Cancer J. Sci. Am. 6(Suppl 1):S61-6; Cao et al., 1998, StemCells 16(Suppl 1):251-60; Kuiper et al., 2000, Adv. Exp. Med. Biol.465:381-90). These molecules can be administered systemically (orlocally) to the host.

A number of means for inducing cellular responses, both in vitro and invivo, are known. Lipids have been identified as agents capable ofassisting in priming CTL in vivo against various antigens. For example,as described in U.S. Pat. No. 5,662,907, palmitic acid residues can beattached to the alpha and epsilon amino groups of a lysine residue andthen linked (for example, via one or more linking residues, such asglycine, glycine-glycine, serine, serine-serine, or the like) to animmunogenic peptide. The lipidated peptide can then be injected directlyin a micellar form, incorporated in a liposome, or emulsified in anadjuvant. As another example, E. coli lipoproteins, such astripalmitoyl-S-glycerylcysteinlyseryl-serine can be used to prime tumorspecific CTL when covalently attached to an appropriate peptide (see,Deres et al., Nature 342:561, 1989). Further, as the induction ofneutralizing antibodies can also be primed with the same moleculeconjugated to a peptide which displays an appropriate epitope, twocompositions can be combined to elicit both humoral and cell-mediatedresponses where that is deemed desirable.

Although administration of VLPs containing an optimized HA protein isexemplified herein, one of skill in the art would understand that it isalso possible to administer the optimized influenza HA protein itself(in the absence of a viral particle) or as a fusion protein to elicit animmune response in a subject.

The following examples are provided to illustrate certain particularfeatures and/or embodiments. These examples should not be construed tolimit the disclosure to the particular features or embodimentsdescribed.

EXAMPLES Example 1 Generation of an H5N1 Influenza COBRA Sequence

This example describes the generation of a H5N1 influenza HA COBRAconsensus sequence using 426 human H5N1 influenza HA sequences fromclades 0, 1, 2.1, 2.2, 2.3 and 7. The resultant COBRA sequence isreferred to as “PATH H5N1 COBRA.”

To generate the final H5N1 influenza HA COBRA sequence, three layers ofconsensus sequences were generated (FIG. 1). In the first layer, sixindividual consensus sequences were generated using (1) 5 clade 0isolates; (2) 86 clade 1 isolates; (3) 106 clade 2.1 isolates; (4) 97clade 2.2 isolates; (5) 34 clade 2.3 isolates; and (6) 1 clade 7isolate. In the second layer, a clade 2 consensus sequence was generatedusing the three clade 2 consensus sequences generated in the first layer(clade 2.1, clade 2.2 and clade 2.3 consensus sequences). A finalconsensus sequence was generated using the individual clade 0, clade 1and clade 7 consensus sequences generated in the first layer and theclade 2 consensus sequence generated in the second layer. The PATH H5N1COBRA sequence is shown below and is set forth herein as SEQ ID NO: 1.

PATH H5N1 COBRA HA (SEQ ID NO: 1)MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKASPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKSSWSNHEASSGVSSACPYQGRSSFFRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTKLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSTEVIKSELEYGNCNTKCQTPMGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQRERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESVKNGTYDYPQYSEEARLNREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLSLWMCSNGSLQCRICI

Example 2 Generation of an H1N1 Influenza COBRA Sequence

This example describes the generation of a H1N1 influenza HA COBRAconsensus sequence using 205 human and swine H1N1 influenza HAsequences. The resultant COBRA sequence is referred to as “PATH H1N1COBRA.”

To generate the final H1N1 influenza HA COBRA sequence, two layers ofconsensus sequences were generated (FIG. 2). In the first layer, fiveindividual consensus sequences were generated using (1) 8 human strainsisolated from 1933-1934; (2) 13 human strains isolated from 1935-1947;(3) 12 human strains isolated from 1948-1957; (4) 123 human strainsisolated from 2009-2011; and (5) 49 swine strains. A final consensussequence was produced using the five individual consensus sequencesgenerated in the first layer. The PATH H1N1 COBRA sequence is shownbelow and is set forth herein as SEQ ID NO: 2.

PATH H1N1 COBRA HA (SEQ ID NO: 2)MKARLLVLLCALAATDADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDSHNGKLCKLKGIAPLQLGKCNIAGWLLGNPECESLLSARSWSYIVETPNSENGTCYPGDFIDYEELREQLSSVSSFERFEIFPKESSWPNHNTTKGVTAACSHAGKSSFYRNLLWLTKKGGSYPKLSKSYVNNKGKEVLVLWGVHHPSTSTDQQSLYQNENAYVSVVSSNYNRRFTPEIAERPKVRGQAGRMNYYWTLLEPGDTIIFEATGNLIAPWYAFALSRGSGSGIITSNASMHECNTKCQTPQGAINSSLPFQNIHPVTIGECPKYVRSTKLRMVTGLRNIPSIQSRGLFGAIAGFIEGGWTGMIDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQFTAVGKEFNNLEKRMENLNKKVDDGFLDIWTYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLRNNAKEIGNGCFEFYHKCDNECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI

Example 3 Codon-Optimized COBRA Gene Sequences

The COBRA amino acid sequences disclosed herein can be reversetranslated and optimized for expression in mammalian cells, includingcodon usage and RNA optimization (GeneArt; Regensburg, Germany). Theoptimized nucleic acid sequences can be inserted into an appropriateexpression vector, such as the pTR600 expression vector (U.S. PatentApplication Publication No. 2002/0106798; Ross et al., Nat Immunol.1(2):102-103, 2000; Green et al., Vaccine 20:242-248, 2001). Expressionvectors encoding the codon-optimized COBRA gene sequences can be used,for example, to generate VLPs containing the COBRA HA.

Example 4 Preparation of and Immunization with Influenza VLPs

The following methods can be used to produce and characterize influenzaVLPs comprising a COBRA HA. Exemplary methods for immunization of mice,ferrets and macaques are also described below (see also, Giles and Ross,Vaccine 29(16):3043-3054, 2011).

Vaccine Preparation

293T cells are transiently transfected with plasmids expressing M1, NAand an optimized HA, and incubated for 72 hours at 37° C. The M1, NA andHA coding sequences can be codon-optimized for expression in mammaliancells. Supernatants are collected and cell debris is removed by lowspeed centrifugation followed by vacuum filtration through a 0.22 μmsterile filter. VLPs are purified via ultracentrifugation (100,000×gthrough 20% glycerol, weight per volume) for 4 hours at 4° C. Thepellets are subsequently resuspended in PBS pH 7.2 and stored in singleuse aliquots at −80° C. until use. Total protein concentration isdetermined by Micro BCA™ Protein Assay Reagent Kit (PierceBiotechnology, Rockford, Ill., USA).

Dose Determination

HA specific content can be determined by western blot and densitometry.Purified recombinant COBRA HA and purified VLPs are prepared in standardtotal protein amounts and are electrophoresed on a 10% SDS-PAGE gel andtransferred to a PVDF membrane. The blot is probed with mouse polyclonalantisera from influenza infected mice and the HA-antibody complexes aredetected using a goat anti-mouse IgG conjugated to horseradishperoxidase (HRP) (Southern Biotech; Birmingham, Ala., USA). HRP isdetected by chemiluminescent substrate (Pierce Biotechnology; RockfordIll., USA) and exposed to X-ray film (ThermoFisher; Pittsburgh, Pa.,USA). Density of bands is determined using ImageJ software (NIH).Density of recombinant HA bands is used to calculate a standard curveand the density of the purified VLPs is interpolated using the resultsfrom the recombinant HA.

Mouse Studies

BALB/c mice (Mus musculus, females, 6-8 weeks old) can be purchased fromHarlan Sprague Dawley (Indianapolis, Ind., USA). Mice are housed inmicroisolator units and allowed free access to food and water and arecared for under USDA guidelines for laboratory animals. Mice arevaccinated with one of three doses of purified COBRA HA VLPs (1.5 μg,0.3 μg or 0.06 μg), based upon HA content from a densitometry assay, viaintramuscular injection at week 0 and then boosted with the same dose atweek 3. Vaccines at each dose are formulated with alum adjuvant (Imject™Alum, Pierce Biotechnology; Rockford, Ill., USA), CpG oligonucleotides,or vehicle alone. Fourteen to twenty-one days after each vaccination,blood is collected from anesthetized mice via the retro-orbital plexusand transferred to a microfuge tube. Tubes are centrifuged and sera isremoved and frozen at −80±5° C. Hemagglutination inhibition (HAI) serumantibody titer for each vaccine group is determined at week 5 usingrepresentative reassortant viruses or COBRA HA VLPs.

Three weeks after final vaccination, mice are challenged intranasallywith a pathogenic H5N1 or H1N1 influenza virus in a volume of 504 Afterinfection, mice are monitored daily for weight loss, disease signs anddeath for 14 days after infection. Individual body weights, sicknessscores (Toapanta and Ross, Respiratory Research 10(1):112, 2009) anddeath are recorded for each group on each day after inoculation.

Ferret Studies

Fitch ferrets (Mustela putorius faro, female, 6-12-months of age),influenza naïve and de-scented, can be purchased from Marshall Farms(Sayre, Pa., USA). Ferrets are pair housed in stainless steel cages(Shor-line, Kansas City, Kans., USA) containing Sani-chips LaboratoryAnimal Bedding (P.J. Murphy Forest Products, Montville, N.J., USA).Ferrets are provided with Teklad Global Ferret Diet (Harlan Teklad,Madison, Wis., USA) and fresh water ad libitum. The COBRA HA VLPs arediluted in PBS, pH 7.2 to achieve final concentration. Ferrets arevaccinated with one of two doses of purified COBRA VLPs (15 μg, 3 μg),based upon HA content as determined by densitometry assay, viaintramuscular injection in the quadriceps muscle in a volume of 0.25 mlat week 0 and then boosted with the same dose at week 3. Vaccines arestored at −80° C. prior to use and formulated with alum adjuvant (ImjectAlum; Pierce Biotechnology, Rockford, Ill., USA) immediately prior touse. Animals are monitored for adverse events including weight loss,temperature, decrease in activity, nasal discharge, sneezing anddiarrhea weekly during the vaccination regimen. Prior to vaccination,animals are confirmed by HAI assay to be seronegative for circulatinginfluenza A and influenza B viruses. Fourteen to twenty-one days aftereach vaccination, blood is collected from anesthetized ferrets via theanterior vena cava and transferred to a microfuge tube. Tubes arecentrifuged and sera is removed and frozen at −80±5° C. HAI serumantibody titer for each vaccine group is determined at week 5 usingrepresentative reassortant viruses or COBRA HA VLPs.

Three weeks after final vaccination, ferrets are challenged intranasallywith a pathogenic H5N1 or H1N1 influenza virus in a volume of 1 ml.After infection, ferrets are monitored daily for weight loss, diseasesigns and death for 14 days after infection. Individual body weights,sickness scores, and death are recorded for each group on each day afterinoculation. Nasal washes are performed by instilling 3 ml of PBS intothe nares of anesthetized ferrets each day for 7 days after inoculation.Washes are collected and stored at −80° C. until use.

Primate Immunizations

Cynomolgus macaques (Macaca fascicularis, male, 3-5 years old) can bepurchased from Harlan Sprague Dawley (Indianapolis, Ind., USA). Macaquesare vaccinated with purified COBRA HA VLPs (15 μg), based upon HAcontent from a densitometry assay, via intramuscular injection at week 0and then boosted with the same dose at weeks 3 and 6. Vaccines areformulated with alum adjuvant (Imject Alum, Pierce Biotechnology;Rockford, Ill., USA) immediately prior to use. Twenty-one days aftereach vaccination, blood is collected from anesthetized macaques via thefemoral vein and transferred to a serum separator tube. Tubes areallowed to activate clotting followed by centrifugation and sera isremoved and frozen at −80±5° C. End point IgG titers and HAI serumantibody titer for each vaccine group is determined at week 5 usingrepresentative reassortant viruses or COBRA HA VLPs.

Three weeks after final vaccination, macaques are challenged byintranasal, intratracheal, and orbital inoculation with a pathogenicH5N1 or H1N1 influenza virus in a volume of 1 ml. After infection,macaques are monitored daily for weight loss, disease signs and deathfor 5 days after infection. Individual body weights, sickness scores anddeath are recorded for each group on each day after inoculation.

Example 5 Immunogenicity and Protective Efficacy of PATH H5N1 COBRAHA-Containing VLPs

This example describes four studies in mice to test the immunogenicityand protective efficacy of PATH H5N1 COBRA VLPs.

COBRA Study 1

This study was conducted to test the immunogenicity and protectiveefficacy of PATH H5N1 COBRA VLPs against clade 1 and clade 2 challenges.VLPs containing a human clade 2 COBRA HA (Human COBRA-2 VLPs) were usedfor comparison.

Mice were vaccinated intramuscularly with 3 μg of VLPs containing thePATH H5N1 COBRA HA sequence (SEQ ID NO: 1), VLPs containing the HumanCOBRA-2 HA sequence, or VLPs containing Whooper Swan (A/WhooperSwan/Mongolia/244/2005) influenza virus HA. Vaccinations were performedat week 0 and week 3 with an adjuvant (Imject™). Mice were challengedwith 5000 PFU of Indonesia clade 2.1 virus (A/Indonesia/5/2005) or 5000PFU of Vietnam clade 1 virus (A/Vietnam/1203/2004) during week 5. Bloodsamples were collected at week 0, week 3 and week 5. Lungs wereharvested at day 3 (D3) after challenge for viral titers.

HAI titers against PATH H5N1 COBRA VLPs (positive control), Vietnamclade 1 virus, Indonesia clade 2.1 virus, Whooper Swam clade 2.2 virus,Egypt/3300/08 clade 2.2.1, Egypt/321/07 clade 2.2.1, Anhui clade 2.3virus and Chicken/Vietnam clade 7 virus in vaccinated mice are shown inFIG. 3. The results demonstrate that vaccination with PATH H5N1 COBRAHA-containing VLPs elicits an antibody response that can recognize bothclade 1 and clade 2 influenza viruses.

Body weights of vaccinated and naïve mice up to day 14 (D14)post-challenge with Indonesia clade 2.1 and with Vietnam clade 1 areshown in FIG. 4 and FIG. 5, respectively. As shown in FIG. 4, the bodyweight of all vaccinated mice changed little over time, whereas naïvemice exhibited a sharp decrease in body weight after day 5 (D5)post-challenge with the clade 2.1 virus. As shown in FIG. 5, the bodyweight of mice vaccinated with PATH H5N1 VLPs did not significantlychange after challenge with the clade 1 virus. In contrast, naïve miceexhibited a rapid weight loss starting at D3 post-challenge.

COBRA Study 2A

This study was conducted to test the protective efficacy against clade 1challenge following a single vaccination of mice with VLPs containingPATH H5N1 COBRA VLPs.

Mice were vaccinated intramuscularly with 3 μg of VLPs containing thePATH H5N1 COBRA HA sequence (SEQ ID NO: 1), VLPs containing the HumanCOBRA-2 HA sequence, or VLPs containing Whooper Swan (A/WhooperSwan/Mongolia/244/2005) influenza virus HA. Vaccination was performed atweek 0 with an adjuvant (Imject™). Mice were challenged with 5000 PFU ofIndonesia clade 2.1 virus (A/Indonesia/5/2005) or 5000 PFU of Vietnamclade 1 virus (A/Vietnam/1203/2004) during week 4. Blood samples werecollected at week 0 and week 3. Lungs were harvested at D2 and D3 afterchallenge for viral titers.

As shown in FIG. 6, the body weight of vaccinated mice did notsignificantly change following challenge with Vietnam clade 1 virus,whereas naïve mice exhibited a significant loss in weight after D3.

COBRA Study 4A

This study was conducted to test protective efficacy against clade 1virus following a single vaccination in the absence of adjuvant. Micewere vaccinated intramuscularly with 3 μg of PATH H5N1 COBRA VLPs, HumanCOBRA-2 VLPs, or Whooper Swan VLPs without adjuvant. Mice werechallenged with 5000 PFU of Vietnam clade 1 virus during week 4. Bloodsamples were collected at week 0 and week 3. Lungs were harvested at D2and D3 after challenge for viral titers.

As shown in FIG. 7, naïve mice succumbed to infection by day 7, whereas40-60% of vaccinated mice survived challenge out to day 14.Specifically, 60% of mice vaccinated with Human COBRA-2 VLPs survivedchallenged while 40% of mice vaccinated with PATH H5N1 COBRA VLPs orWhooper Swan VLPs survived challenge.

COBRA Study 5A

This study was conducted to test protective efficacy against clade 1virus following a single vaccination with adjuvant (Imject™) at a lowerdose of VLP (0.6 μg). Mice were vaccinated intramuscularly with 0.6 μgof PATH H5N1 COBRA VLPs, Human COBRA-2 VLPs, or Whooper Swan VLPs in thepresence of adjuvant. Mice were challenged with 5000 PFU of Vietnamclade 1 virus during week 4. Blood samples were collected at week 0 andweek 3. Lungs were harvested at D2 and D3 after challenge for viraltiters.

As shown in FIG. 8, body weight of vaccinated mice dropped slightlyfollowing virus challenge but returned to normal levels by D10. Incontrast, body weight of naïve mice dropped significantly and the micedid not recover.

In view of the many possible embodiments to which the principles of thedisclosed invention may be applied, it should be recognized that theillustrated embodiments are only preferred examples of the invention andshould not be taken as limiting the scope of the invention. Rather, thescope of the invention is defined by the following claims. We thereforeclaim as our invention all that comes within the scope and spirit ofthese claims.

1. A recombinant influenza hemagglutinin (HA) polypeptide, comprisingthe amino acid sequence of residues 2-566 of SEQ ID NO: 2 or residues2-568 of SEQ ID NO:
 1. 2. The influenza HA polypeptide of claim 1,consisting of the amino acid sequence of residues 2-566 of SEQ ID NO: 2or residues 2-568 of SEQ ID NO:
 1. 3. The influenza HA polypeptide ofclaim 1, comprising the amino acid sequence of SEQ ID NO: 2 or SEQ IDNO:
 1. 4. The influenza HA polypeptide of claim 1, consisting of theamino acid sequence of SEQ ID NO: 2 or SEQ ID NO:
 1. 5. An isolatednucleic acid molecule encoding the influenza HA polypeptide of claim 1.6. The isolated nucleic acid molecule of claim 5, wherein the nucleicacid molecule is codon-optimized for expression in mammalian cells.
 7. Avector comprising the nucleic acid molecule of claim
 5. 8. The vector ofclaim 7, further comprising a promoter operably linked to the nucleicacid sequence encoding the influenza HA polypeptide.
 9. An isolated cellcomprising the vector of claim
 7. 10. An influenza virus-like particle(VLP) comprising the influenza HA polypeptide of claim
 1. 11. Theinfluenza VLP of claim 10, further comprising an influenza neuraminidase(NA) protein, an influenza matrix (M1) protein, or both.
 12. Aninfluenza VLP comprising the influenza HA polypeptide of claim 1,produced by transfecting a host cell with a vector encoding the HApolypeptide, a vector encoding an influenza NA protein and a vectorencoding an influenza M1 protein under conditions sufficient to allowfor expression of the HA, M1 and NA proteins.
 13. A fusion proteincomprising the influenza HA polypeptide of claim
 1. 14. A compositioncomprising the VLP of claim 10 and a pharmaceutically acceptablecarrier.
 15. A method of eliciting an immune response to influenza virusin a subject, comprising administering the VLP of claim
 10. 16. A methodof immunizing a subject against influenza virus, comprisingadministering to the subject a composition comprising the VLP of claim10 and pharmaceutically acceptable carrier.
 17. The method of claim 16,wherein the composition further comprises an adjuvant.
 18. The method ofclaim 16, wherein the composition is administered intramuscularly. 19.The method of claim 16, wherein the composition comprises about 1 toabout 25 μg of the VLP.
 20. The method of claim 19, wherein thecomposition comprises about 15 μg of the VLP.